Humanised antibodies

ABSTRACT

CDR-grafted antibody heavy and light chains comprise acceptor framework and donor antigen binding regions, the heavy chains comprising donor residues at at least one of positions (6, 23) and/or (24, 48) and/or (49, 71) and/or (73, 75) and/or (76) and/or (78) and (88) and/or (91). The CDR-grafted light chains comprise donor residues at at least one of positions (1) and/or (3) and (46) and/or (47) or at at least one of positions (46, 48, 58) and (71). The CDR-grafted antibodies are preferably humanised antibodies, having non human, eg. rodent, donor and human acceptor frameworks, and may be used for in vivo therapy and diagnosis. A generally applicable protocol is disclosed for obtaining CDR-grafted antibodies.

FIELD OF THE INVENTION

The present invention relates to humanized antibody molecules, toprocesses for their production using recombinant DNA technology, and totheir therapeutic uses. The term “humanised antibody molecule” is usedto describe a molecule having an antigen binding site derived from animmunoglobulin from a non-human species, and remainingimmunoglobulin-derived parts of the molecule being derived from a humanimmunoglobulin. The antigen binding site typically comprisescomplementary determining regions (CDRs) which determine the bindingspecificity of the antibody molecule and which are carried onappropriate framework regions in the variable domains. There are 3 CDRs(CDR1, CDR2 and CDR3) in each of the heavy and light chain variabledomains.

In the description, reference is made to a number of publications bynumber. The publications are listed in numerical order at the end of thedescription.

BACKGROUND OF THE INVENTION

Natural immunoglobulin have been known for many years, an have thevarious fragments thereof, such as the Fab, (Fab′)₂ and Fc fragments,which can be derived by enzymatic cleavage. Natural immunoglobulincomprise a generally Y-shaped molecule having an antigen-binding sitetowards the end of each upper arm. The remainder of the structure, andparticularly the stem of the Y, mediates the effector functionsassociated with immunoglobulins.

Natural immunoglobulins have been used in assay, diagnosis and, to amore limited extent, therapy. However, such uses, especially in therapy,were hindered until recently by the polyclonal nature of naturalimmunoglobulins. A significant step towards the realisation of thepotential of immunoglobulins as therapeutic agents was the discovery ofprocedures for the production of monoclonal antibodies (MAbs) of definedspecificity (1).

However, most MAbs are produced by hybridomas which are fusions ofrodent spleen cells with rodent myeloma cells. They are thereforeessentially rodent proteins. There are very few reports of theproduction of human MAbs.

Since most available MAbs are of rodent origin, they are naturallyantigenic in humans and thus can give rise to an undesirable immuneresponse termed the HAMA (Human Anti-Mouse Antibody) response.Therefore, the use of rodent MAbs as therapeutic agents in humans isinherently limited by the fact that the human subject will mount animmunological response to the MAb and will either remove it entirely orat least reduce its effectiveness. In practice, MAbs of rodent originmay not be used in patients for more than one or a few treatments as aHAMA response soon develops rendering the MAb ineffective as well asgiving rise to undesirable reactions. For instance, OKT3 a mouse IgG2a/kNAb which recognizes an antigen in the T-cell receptor-CD3 complex hasbeen approved for use in many countries throughout the world as animmunosuppressant in the treatment of acute allograft rejection[Chatenoud et al (2) and Jeffers et al (3)]. However, in view of therodent nature of this and other such MAbs, a significant HAMA responsewhich may include a major anti-idiotype component, may build up on use.Clearly, it would be highly desirable to diminish or abolish thisundesirable HAMA response and thus enlarge the areas of use of thesevery useful antibodies.

Proposals have therefore been made to render non-human MAbs lessantigenic in humans. Such techniques can be generically termed“humanisation” techniques. These techniques typically involve the use ofrecombinant DNA technology to manipulate DNA sequences encoding thepolypeptide chains of the antibody molecule.

Early methods for humanising MAbs involved production of chimericantibodies in which an antigen binding site comprising the completevariable domains of one antibody is linked to constant domains derivedfrom another antibody. Methods for carrying out such chimerisationprocedures are described in EP0120694 (Celltech Limited), EP0125023(Genentech Inc. and City of Hope), EP-A-0 171496 (Rev. Dev. Corp.Japan), EP-A-0 173 494 (Stanford University), and WO 86/01533 (CelltechLimited). This latter Celltech application (WO 86/01533) discloses aprocess for preparing an antibody molecule having the variable domainsfrom a mouse MAb and the constant domains from a human 4immunoglobulin.Such humanised chimeric antibodies, however, still contain a significantproportion of non-human amino acid sequence, i.e. the complete non-humanvariable domains, and thus may still elicit some HAMA response,particularly if administered over a prolonged period [Begent et al (ref.4)].

In an alternative approach, described in EP-A-0239400 (Winter), thecomplementary determining regions (CDRs) of a mouse MAb have beengrafted onto the framework regions of the variable domains of a humanimmunoglobulin by site directed mutagenesis using long oligonucleotides.The present invention relates to humanised antibody molecules preparedaccording to this alternative approach, i.e. CDR-grafted humanisedantibody molecules. Such CDR-grafted humanised antibodies are much lesslikely to give rise to a HAMA response than humanised chimericantibodies in view of the much lower proportion of non-human amino acidsequence which they contain.

The earliest work on humanising MAbs by CDR-grafting was carried out onMAbs recognizing synthetic antigens, such an the NP or NIP antigens.However, examples in which a mouse MMA recognizing lysozyme and a ratMAb recognizing an antigen on human T-cells were humanised byCDR-grafting have been described by Verhoeyon et al (5) and Riechmann etal (6) respectively. The preparation of CDR-grafted antibody to theantigen on human T calls is also described in WO 89/07452 (MedicalResearch Council).

In Riechmann et al/Medical Research Council it was found that transferof the CDR regions alone [as defined by Kabat refs. (7) and (8)] was notsufficient to provide satisfactory antigen binding activity in theCDR-grafted product. Riechmann et al found that it was necessary toconvert a murine residue at position 27 of the human sequence to thecorresponding rat phenylalanine residue to obtain a CDR-grafted producthaving improved antigen binding activity. This residue at position 27 ofthe heavy chain is within the structural loop adjacent to CDR1. Afurther construct which additionally contained a human serine to rattyrosine change at position 30 of the heavy chain did not have asignificantly altered binding activity over the humanised antibody withthe serine to phenylalanine change at position 27 alone. These resultsindicate that changes to residues of the human sequence outside the CDRregions, in particular in the structural loop adjacent to CDR1, may benecessary to obtain effective antigen binding activity for CDR-graftedantibodies which recognize more complex antigens. Even so the bindingaffinity of the best CDR-grafted antibodies obtained was stillsignificantly less than the original MAb.

Very recently Queen et al (9) have described the preparation of ahumanised antibody that binds to the interleukin 2 receptor, bycombining the CDRs of a marine MAb (anti-Tac) with human immunoglobulinframework and constant regions. The human framework regions were chosento maximise homology with the anti-Tac MAb sequence. In additioncomputer modeling was used to identify framework amino acid residueswhich were likely to interact with the CDR5 or antigen, and mouse Aminoacids were used at these positions in the humanized antibody.

In WO 90/07861 Queen et al propose four criteria for designing humanisedimmunoglobulins. The first criterion is to use as the human acceptor theframework from a particular human immunoglobulin that is unusuallyhomologous to the non-human donor immunoglobulin to be humanised, or touse a consensus framework from many human antibodies. The secondcriterion is to use the donor amino acid rather than the acceptor if thehuman acceptor residue is unusual and the donor residue is typical forhuman sequences at a specific residue of the framework. The thirdcriterion is to use the donor framework amino acid residue rather thanthe acceptor at positions immediately adjacent to the CDRs. The fourthcriterion is to use the donor amino acid residue at framework positionsat which the amino acid is predicted to have a side chain atom withinabout 3 Å of the CDRs in a three-dimensional immunoglobulin model and tobe capable of interacting with the antigen or with the CDRs of thehumanised immunoglobulin. It is proposed that criteria two, three orfour may be applied in addition or alternatively to criterion one, andmay be applied singly or in any combination.

WO 90/07861 describes in detail the preparation of a single CDR-graftedhumanised antibody, a humanised antibody having specificity for the p55Tac protein of the

IL-2 receptor. The combination of all four criteria, as above, wereemployed in designing this humanised antibody, the variable regionframeworks of the human antibody Eu (7) being used as acceptor. In theresultant humanised antibody the donor CDRs were as defined by Kabat etal (7 and 8) and in addition the mouse donor residues were used in placeof the human acceptor residues, at positions 27, 30, 48, 66, 67, 89, 91,94, 103, 104, 105 and 107 in the heavy chain and at positions 48, 60 and63 in the light chain, of the variable region frameworks. The humanisedanti-Tac antibody obtained is reported to have an affinity for pS5 of3×10⁹ M⁻¹, about one-third of that of the murine MAb.

We have further investigated the preparation of CDR-grafted humanisedantibody molecules and have identified a hierarchy of positions withinthe framework of the variable regions (i.e. outside both the Kabat CDRsand structural loops of the variable regions) at which the amino acididentities of the -residues are important for obtaining CDR-graftedproducts with satisfactory binding affinity. This has enabled us toestablish a protocol for obtaining satisfactory CDR-grafted productswhich may be applied very widely irrespective of the level of homologybetween the donor immunoglobulin and acceptor framework. The set ofresidues which we have identified as being of critical importance doesnot coincide with the residues identified by Queen et al (9).

SUMMARY OF THE INVENTION

Accordingly, in a first aspect the invention provides a CDR-graftedantibody heavy chain having a variable region domain comprising acceptorframework and donor antigen binding regions wherein the frameworkcomprises donor residues at at least one of positions 6, 23 and/or 24,48 and/or 49, 71 and/or 73, 75 and/or 76 and/or 78 and 88 and/or 91.

In preferred sediments, the heavy chain framework comprise donorresidues at positions 23, 24, 49, 71, 73 and 78 or at positions 23, 24and 49. The residues at positions 71, 73 and 78 of the heavy chainframework are preferably either all acceptor or all donor residues.

In particularly preferred embodiments the heavy chain frameworkadditionally comprises donor residues at one, some or all of positions6, 37, 48 and 94. Also it is particularly preferred that residues atpositions of the heavy chain framework which are commonly conservedacross species, i.e. positions 2, 4, 25, 36, 39, 47, 93, 103, 104, 106and 107, if not conserved between donor and acceptor, additionallycomprise donor residues. Most preferably the heavy chain frameworkadditionally comprises donor residues at positions 2, 4, 6, 25, 36, 37,39, 47, 48, 93, 94, 103, 104, 106 and 107.

In addition the heavy chain framework optionally comprises donorresidues at one, some or all of positions$

1 and 3,

72 and 76,

69 (if 48 is different between donor and acceptor),

38 and 46 (if 48 is the donor residue),

80 and 20 (if 69 is the donor residue),

67,

82 and 18 (if 67 is the donor residue),

91,

88, and

any one or more of 9, 11, 41, 87, 108, 110 and 112.

In the first and other aspects of the present invention reference ismade to CDR-grafted antibody products comprising acceptor framework anddonor antigen binding regions. It will be appreciated that the inventionis widely applicable to the CDR-grafting of antibodies in general. Thus,the donor and acceptor antibodies may be derived from animals of thesame species and even same antibody class or sub-class. More usually,however, the donor and acceptor antibodies are derived from animals ofdifferent species. Typically the donor antibody is a non-human antibody,such as a rodent MAb, and the acceptor antibody is a human antibody.

In the first and other aspects of the present invention, the donorantigen binding region typically comprises at least one CDR from thedonor antibody. Usually the donor antigen binding region comprises atleast two and preferably all three CDRs of each of the heavy chainand/or light chain variable regions. The CDRs may comprise the KabatCDRs, the structural loop CDRs or a composite of the Kabat andstructural loop CDRs and any combination of any of these. Preferably,the antigen binding regions of the CDR-grafted heavy chain variabledomain comprise CDRs corresponding to the Kabat CDRs at CDR2 (residues50-65) and CDR3 (residues 9S-100) and a composite of the Kabat andstructural loop CDRs at CDR1 (residues 26-35).

The residue designations given above and elsewhere in the presentapplication are numbered according to the Kabat numbering [refs. (7) and(8)5. Thus the residue designations do not always correspond directlywith the linear numbering of the amino acid residues. The actual linearAmino acid sequence may contain fewer or additional amino acids than inthe strict Kabat numbering corresponding to a shortening of, orinsertion into, a structural component, whether framework or CDR, of thebasic variable domain structure. For example, the-heavy chain variableregion of the anti-Tac antibody described by Queen et al (9) contains asingle amino acid insert (residue 52a) after residue 52 of CDR2 and athree amino acid insert (residues 82a, 82b and 82c) after frameworkresidue 82, in the Kabat numbering. The correct Kabat numbering ofresidue may be determined for a given antibody by alignment at regionsof homology of the sequence of the antibody with a standard; Kabatnumbered sequence.

The invention also provides in a second aspect a CDR-grafted antibodylight chain having a variable region domain comprising acceptorframework and donor antigen binding regions wherein the frameworkcomprises donor residues at at least one of positions 1 and/or 3 and 46and/or 47. Preferably the CDR grafted light chain of the second aspectcomprises donor residues at positions 46 and/or 47.

The invention also provides in a third aspect a CDR-grafted antibodylight chain having a variable region domain comprising acceptorframework and donor antigen binding regions wherein the frameworkcomprises donor residues at at least one of positions 46, 48, 58 and 71.

In a preferred embodiment of the third aspects, the framework comprisesdonor residues at all of positions 46, 48, 58 and 71.

In particularly preferred embodiments of the second and third aspects,the framework additionally comprises donor residues at positions 36, 44,47, 85 and 87. Similarly positions of the light chain framework whichare commonly conserved across species, i.e. positions 2, 4, 6, 35, 49,62, 64-69, 98, 99, 101 and 102, if not conserved between donor andacceptor, additionally comprise donor residues. Most preferably thelight chain framework additionally comprises donor residues at positions2, 4, 6, 35, 36, 38, 44, 47, 49, 62, 64-69, 85, 87, 98, 99, 101 and 102.

In addition the framework of the second or third aspects optionallycomprise a donor residues at one, same or all of positions:

1 and 3,

63,

60 (if 60 and 54 are able to form at potential saltbridge),

70 (if 70 and 24 are able to form a potential saltbridge),

73 and 21 (if 47 is different between donor and acceptor),

37 and 45 (if 47 is different between donor and acceptor),

and

any one or more of 10, 12, 40, 80, 103 and 105.

Preferably, the antigen binding regions of the CDR-grafted light chainvariable domain comprise CDRs corresponding to the Kabat CDRs at CDR1(residue 24-34), CDR2 (residues 50-56) and CDR3 (residues 89-97).

The invention further provides in a fourth aspect a CDR-grafted antibodymolecule comprising at least one CDR-grafted heavy chain and at leastone CDR-grafted light chain according to the first and second or firstand third aspects of the invention.

The humanised antibody molecules and chains of the present invention maycomprise: a complete antibody molecule, having full length heavy andlight chains; a fragment thereof, such as a Fab, (Fab′)₂ or FV fragment;a light chain or heavy chain monomer or dimer; or a single chainantibody, e.g. a single chain FV in which heavy and light chain variableregions are joined by a peptide linker; or any other CDR-graftedmolecule with the same specificity as the original donor antibody.Similarly the CDR-grafted heavy and light chain variable region may becombined with other antibody domains as appropriate.

Also the heavy or light chains or humanised antibody molecules of thepresent invention may have attached to them an effector or reportermolecule. For instance, it may have a macrocycle, for chelating a heavymetal atom, or a toxin, such as ricin, attached to it by a covalentbridging structure. Alternatively, the procedures of recombinant DNAtechnology may be used to produce an immunoglobulin molecule in whichthe Fc fragment or CH3 domain of a complete immunoglobulin molecule hasbeen replaced by, or has attached thereto by peptide linkage, afunctional non-immunoglobulin protein, such as an enzyme or toxinmolecule.

Any appropriate acceptor variable region framework sequences may be usedhaving regard to class/type of the donor antibody from which the antigenbinding regions are derived. Preferably, the type of acceptor frameworkused is of the same/similar class/type as the donor antibody.Conveniently, the framework may be chosen to maximise/optimise homologywith the donor antibody sequence particularly at positions close oradjacent to the CDRs. However, a high level of homology between donorand acceptor sequences is not important for application of the presentinvention. The present invention identifies a hierarchy of frameworkresidue positions at which donor residues may be important or desirablefor obtaining a CDR-grafted antibody product having satisfactory bindingproperties. The CDR-grafted products usually have binding affinities ofat least 10⁵ M⁻¹, preferably at least about 10⁸ M⁻¹, or especially inthe range 10⁸-10¹² M⁻¹. In principle, the present invention isapplicable to any combination of donor and acceptor antibodiesirrespective of the level of homology between their sequences. Aprotocol for applying the invention to any particular donor-acceptorantibody pair is given hereinafter. Examples of human frameworks whichmay be used are KOL, NEWM, REI, EU, LAY and POM (refs. 4 and 5) and thelike; for instance KOL and NEWM for the heavy chain and REI for thelight chain and EU, LAY and POM for both the heavy chain and the lightchain.

Also the constant region domains of the products of the invention may beselected having regard to the proposed function of the antibody inparticular the effector functions which may be required. For example,the constant region domains may be human IgA, IgE, IgG or IgM domains.In particular, IgG human constant region domains may be used, especiallyof the IgG1 and IgG3 isotypes, when the humanised antibody molecule isintended for therapeutic uses, and antibody effector functions arerequired. Alternatively, IgG2 and IgG4 isotypes may be used when thehumanised antibody molecule is intended for therapeutic purposes andantibody effector functions are not required, e.g. for simple blockingof lymphokine activity.

However, the remainder of the antibody molecules need not comprise onlyprotein sequences from immunoglobulins. For instance, a gone may beconstructed in which a DNA sequence encoding part of a humanimmunoglobulin chain is fused to a DNA sequence encoding the amino acidsequence of a functional polypeptide such as an effector or reportermolecule.

Preferably the CDR-grafted antibody heavy and light chain and antibodymolecule products are produced by recombinant DNA technology.

Thus in further aspects the invention also includes DNA sequences codingfor the CDR-grafted heavy and light chains, cloning and expressionvectors containing the DNA sequences, host cells transformed with theDNA sequences and processes for producing the CDR-grafted chains andantibody molecules comprising expressing the DNL sequences in thetransformed host cells.

The general methods by which the vectors may be constructed,transfection methods and culture methods are well known per se and forno part of the invention. Such methods are shown, for instance, inreferences 10 and 11.

The DNA sequences which encode the donor amino acid sequence may beobtained by methods well known in the art. For example the donor codingsequences may be obtained by genomic cloning, or cDNA cloning fromsuitable hybridoma cell lines. Positive clones may be screened usingappropriate probes for the heavy and light chain genes in question. AlsoPCR cloning may be used.

DNA coding for acceptor, e.g. human acceptor, sequences may be obtainedin any appropriate way. For example DNA sequences coding for preferredhuman acceptor frameworks such as KOL, REI, EU and NEWM, are widelyavailable to workers in the art.

The standard techniques of molecular biology may be used to prepare DNAsequences coding for the CDR-grafted products. Desired DNA sequences maybe synthesised completely or in part using oligonucleotide synthesistechniques. Site-directed mutagenesis and polymerase chain reaction(PCR) techniques may be used as appropriate. For example oligonucleotidedirected synthesis as described by Jones et al (ref. 20) may be used.Also oligonucleotide directed mutagenesis of a pre-existing variableregion as, for example, described by Verhoeyen et al (ref. 5) orRiechmann et al (ref. 6) may be used. Also enzymatic filling in ofgapped oligonucleotide tides using T4 DNL polymerase as, for exampledescribed by Queen et al (ref. 9) may be used

Any suitable host cell/vector system may be used for expression of theDNA sequences coding for the CDR-grafted heavy and light chains.Bacterial e.g. E. coli, and other microbial systems may be used, inparticular for expression of antibody fragments such as FAb and (Fab′)₂fragments, and especially FV fragments and single chain antibodyfragments e.g. single chain FVs. Eucaryotic e.g. mammalian host cellexpression systems may be used for production of larger CDR-graftedantibody products, including complete antibody molecules. Suitablemammalian host cells include CEO cells and myeloma or hybridoma celllines.

Thus, in a further aspect the present invention provides a process forproducing a CDR-grafted antibody product comprising:

(a) producing in an expression vector an operon having a DNA sequencewhich encodes an antibody heavy chain according to the first aspect ofthe invention;

and/or

(b) producing in an expression vector an operon having a DNA sequencewhich encodes a complementary antibody light chain according to thesecond or third aspect of the invention;

(c) transfecting a host cell with the or each vector; and

(d) culturing the transfected cell line to produce the CDR-graftedantibody product.

The CDR-graft d product may comprise only heavy or light chain derivedpolypeptide, in which case only a heavy chain or light chain polypeptidecoding sequence is used to transfect the host cells.

For production of products comprising both heavy and light chains, thecell line may be transfected with two vectors, the first vector maycontain an operon encoding a light chain-derived polypeptide and thesecond vector containing an operon encoding a heavy chain-derivedpolypeptide. Preferably, the vectors are identical, except in so far asthe coding sequences and selectable markers are concerned, so as toensure as far as possible that each polypeptide chain is equallyexpressed. Alternatively, a single vector may be used, the vectorincluding the sequences encoding both light chain- and heavychain-derived polypeptides.

The DNA in the coding sequences for the light and heavy chains maycomprise cDNA or genomic DNA or both. However, it is preferred that theDNA sequence encoding the heavy or light chain comprises at leastpartially, genomic DNA, preferably a fusion of cDNA and genomic DNA.

The present invention is applicable to antibodies of any appropriatespecificity. Advantageously, however, the invention may be applied tothe humanisation of non-human antibodies which are used for in vivotherapy or diagnosis. Thus the antibodies may be site-specificantibodies such as tumor-specific or cell surface-specific antibodies,suitable for use in in vivo therapy or diagnosis, e.g. tumour imaging.Examples of cell surface-specific antibodies are anti-T cell antibodies,such as anti-CD3, and CD4 and adhesion molecules, such as CR3, ICAM andELAM. The antibodies may have specificity for interleukins (includinglymphokines, growth factors and stimulating factors), hormones and otherbiologically active compounds, and receptors for any of these. Forexample, the antibodies may have specificity for any of the followingsInterferens α, β, γ or , δ, IL1, IL2, IL3, or IL4, etc., TEF, GCSF,GMCSF, EPO, hGH, or insulin, etc.

The the present invention also includes therapeutic and diagnosticcompositions comprising the CDR-grafted products of the invention anduses of such compositions in therapy and diagnosis.

Accordingly in a further aspect the invention provides a therapeutic ordiagnostic composition comprising a CDR-grafted antibody heavy or lightchain or molecule according to previous aspects of the invention incombination with a pharmaceutically acceptable carrier, diluent orexcipient.

Accordingly also the invention provides a method of therapy or diagnosiscomprising administering an effective amount of a CDR-grafted antibodyheavy or light chain or molecule according to previous aspects of theinvention to a human or animal subject.

A preferred protocol for obtaining CDR-grafted antibody heavy and lightchains in accordance with the present invention is set out belowtogether with the rationale by which we have derived this protocol. Thisprotocol and rationale are given without prejudice to the generality ofthe invention as hereinbefore described and defined.

Protocol

It is first of all necessary to sequence the DNA coding for the heavyand light chain variable regions of the donor antibody, to determinetheir amino acid sequences. It is also necessary to choose appropriateacceptor heavy and light chain variable regions, of known amino acidsequence. The CDR-grafted chain is then designed starting from the basisof the acceptor sequence. It will be appreciated that in some cases thedonor and acceptor amino acid residues may be identical at a particularposition and thus no change of acceptor framework residue is required.

1. As a first stop donor residues are substituted for acceptor residuesin the CDRs. For this purpose the CDRs are preferably defined asfollows:

-   -   Heavy chain—CDR1: residues 26-35        -   CDR2: residues 50-65        -   CDR3: residues 95-102    -   Light chain—CDR1: residues 24-34        -   CDR2: residues 50-56        -   CDR3: residues 89-97    -   The positions at which donor residues are to be substituted for        acceptor in the framework are then chosen as follows, first of        all with respect to the heavy chain and subsequently with        respect to the light chain.

2. Heavy Chain

2.1 Choose donor residues at all of positions 23, 24, 49, 71, 73 and 78of the heavy chain or all of positions 23, 24 and 49 (71, 73 and 78 arealways either all donor or all acceptor).

2.2 Check that the following have the same amino acid in donor andacceptor sequences, and if not preferably choose the donor: 2, 4, 6, 25,36, 37, 39, 47, 48, 93, 94, 103, 104, 106 and 107.

2.3 To further optimizes affinity consider choosing donor residues atone, some or any of:

-   -   i. 1, 3    -   ii. 72, 76    -   iii. If 48 is different between donor and acceptor sequences,        consider 69    -   iv. If at 48 the donor residue is chosen, consider 38 and 46    -   v. If at 69 the donor residue is chosen, consider 80 and then 20    -   vi. 67    -   vii. If at 67 the donor residue is chosen, consider 82 and then        18    -   viii. 91    -   ix. 88    -   x. 9, 11, 41, 87, 108, 110, 112

3. Light Chain

3.1 Choose donor at 46, 48, 58 and 71

3.2 Check that the following have the same amino acid in donor andacceptor sequences, if not preferably choose donor:

-   -   2, 4, 6, 35, 38, 44, 47, 49, 62, 64-69 inclusive, 85, 87, 98,        99, 101 and 102

3.3 To further optimise affinity consider choosing donor residues atone, some or any of:

-   -   i. 1, 3    -   ii. 63    -   iii. 60, if 60 and 54 are able to form potential saltbridge    -   iv. 70, if 70 and 24 are able to form potential saltbridge    -   v. 73, and 21 if 47 is different between donor and acceptor    -   vi. 37, and 45 if 47 is different between donor and acceptor    -   vii. 10, 12, 40, 80, 103, 105

Rationale

In order to transfer the binding site of an antibody into a differentacceptor framework, a number of factors need to be considered.

1. The extent of the CDRs

-   -   The CDRs (Complementary Determining Regions) were defined by Wu        and Kabat (refs. 4 and 5) on the basis of an analysis of the        variability of different regions of antibody variable regions.

Three regions per domain were recognised. In the light chain thesequences are 24-34, 50-56, 89-97 (numbering according to Kabat (ref.4), Eu Index) inclusive and in the heavy chain the sequences are 31-35,50-65 and 95-102 inclusive.

-   -   When antibody structures became available it became apparent        that these CDR regions corresponded in the main to loop regions        which extended from the β barrel framework of the light and        heavy variable domains. For H1 there was a discrepancy in that        the loop was from 26 to 32 inclusive and for H2 the loop was 52        to 56and for L2 from 50 to 53. However, with the exception of H1        the CDR regions encompassed the loop regions and extended into        the β strand frameworks. In H1 residue 26 tends to be a serine        and 27 a phenylalanine or tyrosine, residue 29 is a        phenylalanine in most cases. Residues 28 and 30 which are        surface residues exposed to solvent might be involved in        antigen-binding. A prudent definition of the H1 CDR therefore        would include residues 26-35 to include both the loop region and        the hypervariable residues 33-35.    -   It is of interest to note the example of Riechmann et al (ref.        3), who used the residue 31-35 choice for CDR-H1. In order to        produce efficient antigen binding, residue 27 also needed to be        recruited from the donor (rat) antibody.

2. Non-CDR residues which contribute to antigen binding

-   -   By examination of available X-ray structures we have identified        a number of residues which may have an effect on net antigen        binding and which can be demonstrated by experiment. These        residues can be sub-divided into a number of groups.

2.1 Surface residues near CDR (all numbering as in Kabat et al (ref.7)].

2.1.1. Heavy Chain—Key residues are 23, 71 and 73. Other residues whichmay contribute to a lesser extent are 1, 3 and 76. Finally 25 is usuallyconserved but the marine residue should be used if there is adifference.

2.1.2 Light Chain—Many residues close to the CDRs, e.g. 63, 65, 67 and69 are conserved. If conserved none of the surface residues in the lightchain are likely to have a major effect. However, if the murine residueat these positions is unusual, then it would be of benefit to analysethe likely contribution more closely. Other residues which may alsocontribute to binding are 1 and 3, and also 60 and 70 if the residues atthese positions and at 54 and 24 respectively are potentially able toform a salt bridge i.e. 60+54; 70+24.

2.2 Packing residues near the CDRs.

2.2.1. Heavy Chain—Key residues are 24, 49 and 78. Other key residueswould be 36 if not a tryptophan, 94 if not an arginine, 104 and 106 ifnot glycines and 107 if not a throonine. Residues which may make afurther contribution to stable packing of the heavy chain and henceimproved affinity are 2, 4, 6, 38, 46, 67 and 69. 67 packs against theCDR residue 63 and this pair could be either both mouse or both human.Finally, residues which contribute to packing in this region but from alonger range are 18, 20, 90, 82 and 86. 82 packs against 67 and in turn18 packs against 82. 80 packs against 69 and in turn 20 packs against80. 86 forms an B bond network with 38 and 46. Many of the mouse-humandifferences appear minor e.g. Leu-Ile, but could have an minor impact oncorrect packing which could translate into altered positioning of theCDRs.

2.2.2. Light Chain—Key residues are 48, 58 and 71. Other key residueswould be 6 if not glutamine, 35 if not tryptophan, 62 if notphenylalanine or tryosine, 64, 66, 68, 99 and 101 if not glycines and102 if not a threonine. Residues which make a further contribution are2, 4, 37, 45 and 47. Finally residues 73 and 21 and 19 may make longdistance packing contributions of a minor nature.

2.3. Residues at the variable domain interface between heavy and lightchains—In both the light and heavy chains most of the on n-CDR interfaceresidues are conserved. If a conserved residue is replaced by a residueof different character, e.g. size or charge, it should be considered forretention as the murine residue.

2.3.1. Heavy Chain—Residues which need to be considered are 37 if theresidue is not a valine but is of larger side chain volume or has acharge or polarity. Other residues are 39 if not a glutamine, 45 if nota leucine, 47 if not a tryptophan, 91 if not a phenylalanine ortyrosine, 93 if not an alanine and 103 if not a tryptophan. Residue 89is also at the interface but is not in a position where the side chaincould be of great impact.

2.3.2. Light Chain—Residues which need to be considered are 36, if not atyrosine, 38 if not a glutamine, 44 if not a proline, 46, 49 if not atyrosine, residue 85, residue 87 if not a tyrosine and 98 if not aphenylalanine.

2.4. Variable-Constant region interface—The elbow angle between variableand constant regions may be affected by alterations in packing of keyresidues in the variable region against the constant region which mayaffect the position of V_(L) and V_(H) with respect to one another.Therefore it is worth noting the residues likely to be in contact withthe constant region. In the heavy chain the surface residues potentiallyin contact with the variable region are conserved between mouse andhuman antibodies therefore the variable region contact residues mayinfluence the V-C interaction. In the light chain the amino acids foundat a number of the constant region contact points vary, and the V & Cregions are not in such close proximity as the heavy chain. Therefor theinfluences of the light chain V-C interface may be minor.

2.4.1. Heavy Chain—Contact residues are 7, 11, 41, 87, 108, 110, 112.

2.4.2. Light Chain—In the light chain potentially contacting residuesare 10, 12, 40, 80, 83, 103 and 105.

The above analysis coupled with our considerable practical experimentalexperience in the CDR-grafting of a number of different antibodies havelead us to the protocol given above.

The present invention is now described, by way of example only, withreference to the accompanying FIGS. 1-13.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows DNA and amino acid sequences of the OKT3 light chain;

FIG. 2 shows DNA and amino acid sequences of the OKT3 heavy chain;

FIG. 3 shows the alignment of the OKT3 light variable region amino acidsequence with that of the light variable region of the human antibodyREI;

FIG. 4 shows the alignment of the OKT3 heavy variable region amino acidsequence with that of the heavy variable region of the human antibodyKOL;

FIG. 5 shows the heavy variable region amino acid sequences of OKT3, KOLand various corresponding CDR grafts;

FIG. 6 shows the light variable region amino acid sequences of OKT3, REIand various corresponding CDR grafts;

FIG. 7 show a graph of binding assay results for various grafted OKT3antibodies,

FIG. 8 shows a graph of blocking assay results for various grafted OKT3antibodies;

FIG. 9 shows a similar graph of blocking assay results;

FIG. 10 shows similar graphs for both binding assay and blocking assayresults;

FIG. 11 shows further similar graphs for both binding assay and blockingassay results;

FIG. 12 shows a graph of competition assay results for a minimallygrafted OKT3 antibody compared with the OKT3 murine reference standard,and

FIG. 13 shows a similar graph of competition assay results comparing afully grafted OKT3 antibody with the murine reference standard.

DETAILED DESCRIPTION OF EMBODIMENTS OF THE INVENTION EXAMPLE 1CDR-Grafting of OKT3 Materials and Methods

1. Incoming Cells

-   -   Hybridoma cells producing antibody OKT3 were provided by Ortho        (seedlot 4882.1) and were grown up in antibiotic free Dulbecco's        Modified Eagles Medium (DMEM) supplemented with glutamine and 5%        foetal calf serum, and divided to provide both an overgrown        supernatant for evaluation and cells for extraction of RNA. The        overgrown supernatant was shown to contain 250 ug/mL murine        IgG2a/kappa antibody. The supernatant was negative for murine        lambda light chain and IgG1, IgG2b, IgG3, IgA and IgM heavy        chain. 20 mL of supernatant was assayed to confirm that the        antibody present was OKT3.

2. Molecular Biology Procedures

-   -   Basic molecular biology procedures were as described in Maniatis        et al (ref. 9) with, in some cases, minor modifications. DNA        sequencing was performed as described in Sanger et al (ref. 11)        and the Amersham International Plc sequencing handbook. Site        directed mutagenesis was as described in Kramer et al (ref. 12)        and the Anglian Biotechnology Ltd. handbook. COS cell expression        and metabolic labelling studies were as described in Whittle et        al (ref. 13)

3. Research Assays

3.1. Assembly Assays

-   -   Assembly assays were performed on supernatants from transfected        COS cells to determine the amount of intact IgG present.

3.1.1. COS Cells Transfected with Mouse OKT3 Genes

-   -   The assembly assay for intact mouse IgG in COS cell supernatants        was an ELISA with the following formats    -   96 well microtitre plates were coated with Of(ab′)2 goat        anti-mouse IgG Fc. The plates were washed in water and samples        added for 1 hour at room temperature. The plates were washed and        Of(ab′)2 goat anti-mouse IgG P(ab′)2 (ERPO conjugated) was then        added. Substrate was added to reveal the reaction. UPC10, a        mouse IgG2a myeloma, was used as a standard. 3.1.2. COS and CHO        Cells Transfected with Chimeric or CDR-Grafted OKT3 Genes    -   The assembly assay for chimeric or CDR-grafted antibody in COS        cell supernatants was an ELISA with the following-format:    -   96 well microtitre plates were coated with Of(ab′)2 goat        anti-human IgG Fc. The plates were washed and samples added and        incubated for 1 hour at room temperature. The plates were washed        and monoclonal mouse anti-human kappa chain was added for 1 hour        at room temperature.    -   The plates were washed and Of(ab′)2 goat anti-mouse IgG Fc (ERPO        conjugated) was added. Enzyme substrate was added to reveal the        reaction. Chimeric B72.3 (IgG4) (ref. 13) was used as a        standard. The use of a monoclonal anti-kappa chain in this assay        allows grafted antibodies to be read from the chimeric standard.

3.2. Assay for Antigen Binding Activity

-   -   Material from COS cell supernatants was assayed for OKT3 antigen        binding activity onto CD3 positive cells in a direct assay. The        procedure was as follows:    -   HUT 78 cells (human T cell line, CD3 positive) were maintained        in culture. Monolayers of EUT 78 calls were prepared onto 96        well ELISA plates using poly-L-lyine and glutaraldehyde. Samples        were added to the monolayers for 1 hour at room temperature.    -   The plates were washed gently using PBS. Of(ab′)2 goat        anti-human IgG Fc (ERPO conjugated) or Of(ab′)2 goat anti-mouse        IgG Fc (ERPO conjugated) was added as appropriate for humanised        or mouse samples. Substrate was added to reveal the reaction.    -   The negative control for the cell-based assay was chimeric        B72.3. The positive control was mouse Orthomune OKT3 or chimeric        OKT3, when available. This cell-based assay was difficult to        perform, and an alternative assay was developed for CDR-grafted        OKT3 which was more sensitive and easier to carry out.    -   In this system CDR-grafted OKT3 produced by COS cells was tested        for its ability to bind to the CD3-positive EPB-ALL (human        peripheral blood acute lymphocytic leukemia) cell line. It was        also tested for its ability to block the binding of murine OKT3        to these cells. Binding was measured by the following procedure:        HPB-ALL cells were harvested from tissue culture. Cells were        incubated at 4° C. for 1 hour with various dilutions of test        antibody, positive control antibody, or negative control        antibody. The cells were washed once and incubated at 4° C. for        1 hour with an FITC-labelled goat anti-human IgG (Fc-specific,        mouse absorbed). The cells were washed twice and analysed by cyt        fluorography. Chimeric OKT3 was used as a positive control for        direct binding. Cells incubated with mock-transfected COS call        supernatant, followed by the FITC-labelled goat anti-human IgG,        provided the negative control. To test the ability of        CDR-grafted OKT3 to block murine OKT3 binding, the HPB-ALL calls        were incubated at 4° C. for 1 hour with various dilutions of        test antibody or control antibody. A fixed saturating amount of        FITC OKT3 was added. The samples were incubated for 1 hour at 4°        C., washed twice and analysed by cyt of fluorography.        FITC-labelled OKT3 was used as a positive control to determine        maximize binding. Unlabelled murine OKT3 served as a reference        standard for blocking. Negative controls were unstained cells        with or without mock-transfected cell supernatant. The ability        of the CDR-grafted OKT3 light chain to bind CD3-positive cells        and block the binding of murine OKT3 was initially tested in        combination with the chimeric OKT3 heavy chain. The chimeric        OKT3 heavy chain is composed of the murine OKT3 variable region        and the human IgG constant region. The chimeric heavy chain gene        is expressed in the same expression vector used for the        CDR-grafted genes. The CDR-grafted light chain expression vector        and the chimeric heavy chain expression vector were        co-transfected into COS cells. The fully chimeric OKT3 antibody        (chimaoic light chain and chimeric heavy chain) was found to be        fully capable of binding to CD3 positive cells and blocking the        binding of murine OKT3 to these cells.

3.3 Determination of Relative Binding Affinity

-   -   The relative binding affinities of CDR-grafted anti-CD3        monoclonal antibodies were determined by competition binding        (ref. 6) using the HPB-ALL human T cell line as a source of CD3        antigen, and fluorescein-conjugated murline OKT3 (Fl-OKT3) of        known binding affinity as a tracer antibody. The binding        affinity of Fl-OKT3 tracer antibody was determined by a direct        binding assay in which increasing amounts ad Fl-OKT3 were        incubated with HPB-ALL (5×10⁵) in PBS with 5% foetal calf serum        for 60 min. at 4° C. Cells were washed, and the fluorescence        intensity was determined on a FACScan flow cytometer calibrated        with quantitative microbead standards (Flow Cytometry Standards,        Research Triangle Park, N.C.). Fluorescence intensity per        antibody molecule (Of/P ratio) was determined by using        microbeads which have a predetermined number of mouse IgG        antibody binding sites (Simply Cellular beads, Flow Cytometry        Standards). Of/P equals the fluorescence intensity of beads        saturated with Fl-OKT3 divided by the number of binding sites        per bead. The amount of bound and free Fl-OKT3 was calculated        from the mean fluorescence intensity per cell, and the ratio of        bound/free was plotted against the number of moles of antibody        bound. A linear fit was used to determine the affinity of        binding (absolute value of the slope).    -   For competitive binding, increasing amounts of competitor        antibody were added to a sub-saturating dose of Fl-OKT3 and        incubated with 5×10⁵ HPB-ALL in 200 ml of PBS with 5% foetal        calf serum, for 60 min at 4° C. The fluorescence intensities of        the cells were measured on a FACScan flow cytometer calibrated        with quantitative microbead standards The concentrations of        bound and free Fl-OKT3 were calculated. The affinities of        competing anti-bodies were calculated from the equation        [X]−[OKT3]=(1/Kx)−(1/Ka), where Ka is the affinity of murine        TO3, Kx is the affinity of competitor X, [ ] is the        concentration of competitor antibody at which bound/free binding        is R/2, and R in the maximal bound/free binding.

4. cDNA Library Construction

4.1. mRNA Preparation and cDNA Synthesis

-   -   OKT3 producing cells were grown as described above and 1.2×10⁹        cells harvested and mRNA extracted using the guanidinium/LiCl        extraction procedure. cDNA was prepared by priming from Oligo-dT        to generate full length cDNA. The cDNA was methylated and EcoRl        linkers added for cloning.

4.2. Library Construction

-   -   The cDNA library was ligated to pSP65 vector DNA which had been        EcoRl cut and the 5′ phosphate groups removed by calf intestinal        phosphatase (EcoRl/CIP). The ligation was used to transform high        transformation efficiency Escherichia coli (E. coli) HEB101. A        cDNA library was prepared. 3600 colonies were screened for the        light chain and 10000 colonies were screened for the heavy        chain.

5. Screening

-   -   E. coli colonies positive for either heavy or light chain probes        were identified by oligonucleotide screening using the        oligonucleotides:    -   5′ TCCAGATGTTAACTGCTCAC for the light chain, which is        complementary to a sequence in the mouse kappa constant region,        and 5′ CAGGGGCCAGTGGATGGATAGAC for the heavy chain which is        complementary to a sequence in the mouse IgG2a constant CH1        domain region. 12 light chain and 9 heavy chain clones were        identified and taken for second round screening. Positive clones        from the second round of screening were grown up and DNA        prepared. The sizes of the gene inserts were estimated by gel        electropharesis and inserts of a size capable of containing a        full length cDNA were subcloned into M13 for DNA sequencing.

6. DNA Sequencing

-   -   Clones representing four size classes for both heavy and light        chains were obtained in M13. DNA sequence for the 5′        untranslated regions, signal sequences, variable regions and 3′        untranslated regions of full length cDNAs [FIGS. 1(a) and 2(a)]        were obtained and the corresponding amino acid sequences        predicted [(FIGS. 1(b) and 2(b)]. In FIG. 1(a) the untranslated        DNA regions are shown in uppercase, and in both FIGS. 1 and 2        the signal sequences are underlined.

7. Construction of cDNA Expression Vectors

-   -   Celltech expression vectors are based on the plasmid pEE6hCMV        (ref. 14). A polylinker for the insertion of genes to be        expressed has been introduced after the major immediate early        promoter/enhancer of the human Cytomegalovirus (hCMV). Marker        genes for selection of the plasmid in transfected eukaryotic        cells can be inserted as BlmHl cassettes in the unique BamHl        site of pEE6 hCMV; for instance, the neo marker to provide pEE6        hCMV neo. It is usual practice to insert the neo and gpt markers        prior to insertion of the gene of interest, whereas the GS        marker is inserted last because of the presence of internal        EcoRl sites in the cassette.    -   The selectable markers are expressed from the SV40 late promoter        which also provides an origin of replication so that the vectors        can be used for expression in the COS cell transient expression        system.    -   The mouse sequences were excised from the M13 based vectors        described above as EcoRl fragments and cloned into either        pBE6-hCMV-neo for the heavy chain and into EZ6-hCMV-gpt for the        light chain to yield vectors pJA136 and pJA135 respectively.

8. Expression of cDNAS in COS Cells

-   -   Plasmids pJA135 and pJA136 were co-transfected into COS cells        and supernatant from the transient expression experiment was        shown to contain assembled antibody which bound to T-cell        enriched lymphocytes. Metabolic labelling experiments using ³⁵S        methionine showed expression and assembly of heavy and light        chains.

9. Construction of Chimeric Genes

-   -   Construction of chimeric genes followed a previously described        strategy [Whittle et al (ref. 13)]. A restriction site near the        3′ end of the variable domain sequence is identified and used to        attach an oligonucleotide adapter coding for the reminder of the        mouse variable region and a suitable restriction site for        attachment to the constant region of choice.

9.1. Light Chain Gene Construction

The mouse light chain cDNA sequence contains an Aval site near the 3′end of the variable region [FIG. 1(a)]. The majority of the sequence ofthe variable region was isolated as a 396 bp. EcoRl-Aval fragment. Anoligonucleotide adapter was designed to replace the remainder of the 3′region of the variable region from the Aval sit and to include the 5′residues of the human constant region up to and including a unique Narlsite which had been previously engineered into the constant region.

-   -   A Hindlll site was introduced to act as a marker for insertion        of the linker.    -   The linker was ligated to the V_(L) fragment and the 413 bp        EcoRl-Narl adapted fragment was purified from the ligation        mixture.    -   The constant region was isolated as an Narl-BamHl fragment from        an M13 clone NW361 and was ligated with the variable region DNA        into an EcoRl/BamHl/ClP pSP65 treated vector in a three way        reaction to yield plasmid JA143. Clones were isolated after        transformation into E. coli and the linker and junction        sequences were confirmed by the presence of the Hindlll site and        by DNA sequencing.

9.2 Light Chain Gene Construction—Version 2

The construction of the first chimeric light chain gene produces afusion of mouse and hu amino acid sequences at the variable-constantregion junction. In the case of the OKT3 light chain the amino acids atthe chimera junction are:........Leu-Glu-Ile-Asn-Arg/  -/Thr-Val-Ala   -Ala                    VARIABLE        CONSTANT

-   -   This arrangement of sequence introduces a potential site for        Asparagine (Asn) linked (N-linked) glycosylation at the V-C        junction. Therefore, a second version of the chimeric light        chain oligonucleotide adapter was designed in which the        threonine (Thr), the first amino acid of the human constant        region, was replaced with the equivalent amino acid from the        mouse constant region, Alanine (Ala).    -   An internal Hindlll site was not included in this adapter, to        differentiate the two chimeic light chain genes.    -   The variable region fragment was isolated as a 376 bp EcoRl-Aval        fragment. The aligonucleotide linker was ligated to Narl cut        pNW361 and then the adapted 396bp constant region was isolated        after recutting the modified pNW361 with EcoRl. The variable        region fragment and the modified constant region fragment were        ligated directly into EcoRl/ClP treated pEZ6hCMVneo to yield        pJA137. Initially all clones examined had the insert in the        incorrect orientation. Therefore, the insert was re-isolated and        recloned to turn the insert round and yield plamid pJA141.        Several clones with the insert in the correct orientation were        obtained and the adapter sequence of one was confirmed by DNA        sequencing

9.3. Heavy Chain Gene Construction

9.3.1. Choice of Heavy Chain Gene Isotype

-   -   The constant region isotype chosen for the heavy chain was human        IgG4.

9.3.2. Gene Construction

-   -   The heavy chain cDNA sequence showed a BanI site near the 3′ end        of the variable region [FIG. 2(a)]. The majority of the sequence        of the variable region was isolated as a 426 bp. EcoRl/ClP/Banl        fragment. An oligonucleotide adapter was designated to replace        the remainder of the 3′ region of the variable region from the        Banl site up to and including a unique HindIII site which had        been previously engineered into the first two amino acids of the        constant region.    -   The linker was ligated to the V_(H) fragment and the        EcoRl-Hindlll adapted fragment was purified from the ligation        mixture.

The variable region was ligated to the constant region by cutting pJA91with EcoRl and Hindlll removing the intron fragment and replacing itwith the V_(H) to yield pJA142. Clones were isolated aftertransformation into E. coli JM101 and the linker and junction sequenceswere confirmed by DNA sequencing. (N.B. The Hindlll site in lost oncloning).

10. Construction of Chimeric Expression Vectors

10.1. neo AND gpt Vectors

-   -   The chimeric light chain (version 1) was removed from pJA143 as        an EcoRl fragment and cloned into EcoRl/ClP treated pEE6hCMVneo        expression vector to yield pJA145. Clones with the insert in the        correct orientation were identified by restriction mapping.    -   The chimeric light chain (version 2) was constructed as        described above.    -   The chimeric heavy chain gene was isolated from pJA142 as a        2.5SKbp EcoRl/BaHl fragment and cloned into the EcoRl/Bcll/ClP        treated vector fragment of a derivative of pEE6hCMVgpt to yield        plasmid pJA144.    -   10.2. GS Separate Vectors    -   GS versions of pJA141 and pJA144 were constructed by replacing        the neo and gpt cassettes by a BamHl/Sall/ClP treatment of the        plasmids, isolation of the vector fragment and ligation to a        GS-containing fragment from the plasmid pRO49 to yield the light        chain vector pJA179 and the heavy chain vector pJA180.

10.3. GS Single Vector Construction

-   -   Single vector constructions containing the cL (chimeric light),        cE (chimeric heavy) and GS genes on one plasmid in the order        cL-cH-GS, or cH-cL-GS and with transcription of the genes being        head to tail e.g. cL>c S were constructed. These plasmids were        made by treating pJA179 or pJA180 with BamHl/ClP and ligating in        a Bglll/Hindlll hCMV promoter cassette along with either the        Hindlll/BamHl fragment from pJA141 into pJA180 to give the        cH-cL-GW plasmid pJA182 or the Hindlll/BamHl fragment from        pJA144 into pJA179 to give the cL-cg-GS plasmid pJA181.

11. Expression of Chimeric Genes

11.1. Expression in COS Cells

-   -   The chimeric antibody plasmid pJA145 (cL) and pJA144 (cH) were        co-transfected into COS cells and supernatant from the transient        expression experiment was shown to contain assembled antibody        which bound to the HUT 78 human T-cell line. Metabolic labelling        experiments using ³⁵S methionine showed expression and assembly        of heavy and light chains. However the light chain mobility seen        on reduced gels suggested that the potential glycosylation site        was being glycosylated. Expression in COS cells in the presence        of tunicamycin showed a reduction in size of the light chain to        that shown for control chimeric antibodies and the OKT3 mouse        light chain. Therefore JA141 was constructed and expressed. In        this case the light chain did not show an aberrant mobility or a        size shift in the presence or absence of tunicamycin. This        second version of the chimeric light chain, when expressed in        association with chimeric heavy (cH) chain, produced antibody        which showed good binding to HUT 78 cells. In both cases antigen        binding was equivalent to that of the mouse antibody.

11.2 Expression in Chinese Hamster Ovary (CEO) Cells

-   -   Stable cell lines have been prepared from plasmids PJA141/pJA144        and from pJA179/pJA180, pJA181 and pJA182 by transfection into        CEO cells.

12. CDR-Grafting

-   -   The approach taken was to try to introduce sufficient mouse        residues into a human variable region framework to generate        antigen binding activity comparable to the mouse and chimeric        antibodies.

12.1. Variable Region Analysis

-   -   From an examination of a small database of structures of        antibodies and antigen-antibody complexes it is clear that only        a small number of antibody residues make direct contact with        antigen. Other residues may contribute to antigen binding by        positioning the contact residues in favourable configurations        and also by inducing a stable packing of the individual variable        domains and stable interaction of the light and heavy chain        variable domains. The residues chosen for transfer can be        identified in a number of ways:    -   (a) By examination of antibody X-ray crystal structures the        antigen binding surface can be predominantly located on a series        of loops, three per domain, which extend from the B-barrel        framework.    -   (b) By analysis of antibody variable domain sequences regions of        hypervariability [termed the Complementary Determining Regions        (CDRs) by Wu and Kabat (ref. 5)] can be identified. In the most        but not all cases these CDRs correspond to, but extend a short        way beyond, the loop regions noted above.    -   (c) Residues not identified by (a) and (b) may contribute to        antigen binding directly or indirectly by affecting antigen        binding site topology, or by inducing a stable packing of the        individual variable domains and stabilising the inter-variable        domin interaction. These residues may be identified either by        superimposing the sequences for a given antibody on a known        structure and looking at key residues for their contribution, or        by sequence alignment analysis and noting “idiosyncratic”        residues followed by examination of their structural location        and likely effects.

12.1.1. Light Chain

-   -   FIG. 3 shows an alignment of sequences for the human framework        region RE1 and the OKT3 light variable region. The structural        loops (LOOP) and CDRs (KT) believed to correspond to the antigen        binding region are marked. Also marked are a number of other        residues which may also contribute to antigen binding as        described in 13.1(c). Above the sequence in FIG. 3 the residue        type indicates the spatial location of each residue side chain,        derived by examination of resolved structures from X-ray        crystallography analysis. The key to this residue type        designation is as follows:    -   N—near to CDR (From X-ray Structures)    -   P—Packing B—Buried Non-Packing    -   S—Surface B—Exposed    -   I—Interface *—Interface    -   —Packing/Part Exposed    -   ?—Non-CDR Residues which may require to be left as Mouse        sequence.    -   Residues underlined in FIG. 3 are amino acids. RE1 was chosen as        the human framework because the light chain is a kappa chain and        the kappa variable regions show higher homology with the mouse        sequences than a lambda light variable region, e.g. KOL (see        below). RP1 was chosen in preference to another kappa light        chain because the X-ray structure of the light chain has been        determined so that a structural examination of individual        residues could be made.

12.1.2. Heavy Chain

-   -   Similarly FIG. 4 shows an alignment of sequences for the human        framework region KOL and the OKT3 heavy variable region. The        structural loops and CDRs believed to correspond to the antigen        binding region are marked. Also marked are a number of other        residues which may also contribute to antigen binding as        described in 12.1(c). The residue type key and other indicators        used in FIG. 4 are the same as those used in FIG. 3. KOL was        chosen as the heavy chain framework because the X-ray structure        has been determined to a better resolution than, for example,        NEWM and also the sequence alignment of OKT3 heavy variable        region showed a slightly better homology to KOL than to NEWM.

12.2. Design of Variable Genes

-   -   The variable region domains were designed with mouse variable        region optimal codon usage [Grantham and Perrin (ref. 15)] and        used the B72.3 signal sequences [Whittle et al (ref. 13)]. The        sequences were designed to be attached to the constant region in        the same way as for the chimeric genes described above. Some        constructs contained the “Kozak consensus sequence” [Kozak (ref.        16)] directly linked to the 5′ of the signal sequence in the        gene. This sequence motif is believed to have a beneficial role        in translation initiation in eukaryotes.

12.3. Gene Construction

-   -   To build the variable region, various strategies are available.        The sequence may be assembled by using oligonuleotides in a        manner similar to Jones et al (ref. 17) or by simultaneously        replacing all of the CDRs or loop regions by oligonucleotide        directed site specific mutagenesis in a manner similar to        Verhoeyen et al (ref. 2). Both strategies were used and a list        of constructions is set out in Tables 1 and 2 and FIGS. 4 and 5.        It was noted in several cases that the mutagenesis approach led        to deletions and rearrangements in the gene being remodelled,        while the success of the assembly approach was very sensitive to        the quality of the oligonucleotides.

13. Construction of Expression Vectors

Genes were isolated from M13 or SP65 based intermediate vectors andcloned into pEE6hCXVneo for the light chains and pEE6hCMVgpt for theheavy chains in a manner similar to that for the chimeric genes asdescribed above. TABLE 1 CDR-GRAFTED GENE CONSTRUCTS KOZAK METHOD OFSEQUENCE CODE MOUSE SEQUENCE CONTENT CONSTRUCTION − + LIGHT CHAIN ALLHUMAN FRAMEWORK RE1 121 26-32, 50-56, 91-96 inclusive SDM and geneassembly + n.d. 121A 26-32, 50-56, 91-96 inclusive Partial gene assemblyn.d. + +1, 3, 46, 47 121B 26-32, 50-56, 91-96 inclusive Partial geneassembly n.d. + +46, 47 221 24-24, 50-56, 91-96 inclusive Partial geneassembly + + 221A 24-34, 50-56, 91-96 inclusive Partial geneassembly + + +1, 3, 46, 47 221B 24-34, 50-56, 91-96 inclusive Partialgene assembly + + +1, 3 221C 24-34, 50-56, 91-96 inclusive Partial geneassembly + + HEAVY CHAIN ALL HUMAN FRAMEWORK KOL 121 26-32, 50-56,95-100B inclusive Gene assembly n.d. + 131 26-32, 50-58, 95-100Binclusive Gene assembly n.d. + 141 26-32, 50-65,95-100B inclusivePartial gene assembly + n.d. 321 26-35, 50-56, 95-100B inclusive Partialgene assembly + n.d. 331 26-35, 50-58, 95-100B inclusive Partial geneassembly + Gene assembly + 341 26-35, 50-65, 95-100B inclusive SDM +Partial gene assembly + 341A 26-35, 50-65, 95-100B inclusive Geneassembly n.d. + +6, 23, 24, 48, 49, 71, 73, 76, 78, 88, 91 (+63 = human)341B 26-35, 50-65, 95-100B inclusive Gene assembly n.d. + +48, 49, 71,73, 76, 78, 88, 91 (+63 + human) KEY n.d. not done SDM Site directedmutagenesis Gene assembly Variable region assembled entirely fromoligonucleotides Partial gene Variable region assembled by combinationof restriction assembly fragments either, from other genes originallycreated by SDM and gene assembly or by oligonucleotide assembly of partof th variable region and rec nstruction with restriction fragments fromother genes originally created by SDM and gene assembly

14. Expression of CDR-Grafted Genes

14.1. Production of Antibody Consisting of Grafted Light (gL) Chainswith Mouse Heavy (mH) or Chimeric Heavy (cH) Chains

-   -   All gL chains, in association with mH or cH produced reasonable        amounts of antibody. Insertion of the Kozak consensus sequence        at a position 5′ to the ATG (kgL constructs) however, led to a        2-5 fold improvement in net expression. Over an extended series        of experiments expression levels were raised from approximately        200 ng/ml to approximately 500 ng/ml for kgL/cH or kgL/mH        combinations.    -   When direct binding to antigen on HUT 78 cells was measured, a        construct designed to include mouse sequence based on loop        length (gL121) did not lead to active antibody in association        with mH or cH. A construct designed to include mouse sequence        based on Kabat CDRs (gL221) demonstrated some weak binding in        association with mH or cH. However, when framework residues 1,        3, 46, 47 were changed from the human to the murine OKT3        equivalents based on the arguments outlined in Section 12.1        antigen binding was demonstrated when both of the new        constructs, which were termed 121A and 221A were co-expressed        with cH. When the effects of these residues were examined in        more detail, it appears that residues 1 and 3 are not major        contributing residues as the product of the gL221B gene shows        little detectable binding activity in association with cH. The        light chain product of gL221C, in which mouse sequences are        present at 46 and 47, shows good binding activity in association        with cH.

14.2 Production of Antibody Consisting of Grafted Heavy (gH) Chains withMouse Light (mL) or Chimeric Light (cL) Chains

-   -   Expression of the gH genes proved to be more difficult to        achieve than for gL. First, inclusion of the Kozak sequence        appeared to have no marked effect on expression of gH genes.        Expression appears to be slightly improved but not to the same        degree as seen for the grafted light chain.    -   Also, it proved difficult to demonstrate production of expected        quantities of material when the loop choice (amino acid 26-32)        for CDR1 is used, e.g. gH121, 131, 141 and no conclusions can be        drawn about these constructs.    -   Moreover, co-expression of the gH341 gene with cL or mL has been        variable and has tended to produce lower amounts of antibody        than the cH/cL or mH/mL combinations. The alterations to gH341        to produce gH341A and gH341B lead to improved levels of        expression.    -   This may be due either to a general increase in the fraction of        mouse sequence in the variable region, or to the alteration at        position 63 where the residue is returned to the human amino        acid Valine (Val) from Phenylalanine (Phe) to avoid possible        internal packing problems with the rest of the human framework.        This arrangement also occurs in gH331 and gH321.    -   When gH321 or gH331 were expressed in association with cL,        antibody was produced but antibody binding activity was not        detected. When the more conservative gH341 gene was used antigen        binding could be detected in association with cL or mL, but the        activity was only marginally above the background level.    -   When further mouse residues were substituted based an the        arguments in 12.1, antigen being could be clearly demonstrated        for the antibody produced when kgH341A and kgH341B were        expressed in association with cL.

14.3 Production of Fully CDR-Grafted Antibody

-   -   The kgL221L gene was co-expressed with kgB341, kgH341A or        kgH341B. For the combination kgH221A/kgH341 very little material        was produced in a normal COS cell expression.    -   For the combinations kgL221A/kgH341A or kH221A/kgH341B amounts        of antibody similar to gL/cH was produced.    -   In several experiments no antigen binding activity could be        detected with kgH221A/gH341 or kgH221A/kgH341 combinations,        although expression levels were very low.    -   Antigen binding was detected when kgL221A/kgH341A or        kgH221A/kgH341B combinations were expressed. In the case of the        antibody produced from the kgL221A/kgH341A combination the        antigen binding was very similar to that of the chimeric        antibody.    -   An analysis of the above results is given below.

15. Discussion of CDR-Grafting Results

-   -   In the design of the fully humanised antibody the aim was to        transfer the minimum number of mouse amino acids that would        confer antigen binding onto a human antibody framework.

15.1. Light Chain

15.1.1 Extent of the CDRs

-   -   For the light chain the regions defining the loops known from        structural studies of other antibodies to contain the antigen        contacting residues, and those hypervariable sequences defined        by Kabat et al (refs. 4 and 5) as Complementary Determining        Regions (CDRs) are equivalent for CDR2. For CDR1 the        hypervariable region extends from residues 24-34 inclusive while        the structural loop extends from 26-32 inclusive. In the case of        OKT3 there is only one amino acid difference between the two        options, at Amino acid 24, where the mouse sequence is a serine        and the human framework RE1 has glutamine. For CDR3 the loop        extends from residues 91-96 inclusive while the Kabat        hypervariability extends from residues 89-97 inclusive. For OKT3        amino acids 89, 90 and 97 are the same between OKT3 and RE1        (FIG. 3). When constructs based on the loop choice for CDR1        (gL121) and the Kabat choice (gL221) were made and co-expressed        with mH or cH no evidence for antigen binding activity could be        found for gL121, but trace activity could be detected for the        gL221, suggesting that a single extra mouse residue in the        grafted variable region could have some detectable effect. Both        gene constructs were reasonably well expressed in the transient        expression system.

15.1.2. Framework Residues

-   -   The remaining framework residues were then further examined, in        particular amino acids known from X-ray analysis of other        antibodies to be close to the CDRs and also those amino acids        which in OKT3 showed differences from the consensus framework        for the mouse subgroup (subgroup VI) to which OKT3 shows most        homology. Four positions 1, 3, 46 and 47 were identified and        their possible contribution was examined by substituting the        mouse amino acid for the human amino acid at each position.        Therefore gL221A (gL221+D1Q, Q3V, L46R, L47W, see FIG. 3 and        Table 1) was made, cloned in EE6hCMVneo and co-expressed with cH        (PJA144). The resultant antibody was well expressed and showed        good binding activity. When the related genes gL221B (gL221        +DlQ, Q3V) and gL221C (gL221+L46R, L47W) were made and similarly        tested, while both genes produced antibody when co-expressed        with cH, only the gL221C/cH combination showed good antigen        binding. When the gL121A (gL121+D1Q, Q3V, L46R, L47W) gene was        made and co-expressed with cH, antibody was produced which also        bound to antigen.

15.2. Heavy Chain

15.2.1. Extent of the CDRs

-   -   For the heavy chain the loop and hypervariability analyses agree        only in CDR3. For CDR1 the loop region extends from residues        26-32 inclusive whereas the Kabat CDR extends from residues        31-35 inclusive. For CDR2 the loop region is from 50-56        inclusive while the hypervariable region covers amino acids        50-65 inclusive. Therefore humanised heavy chains were        constructed using the framework from antibody KOL and with        various combinations of these CDR choices, including a shorter        choice for CDR2 of 50-56 inclusive as there was some uncertainty        as to the definition of the end point for the CDR2 loop around        residues 56 to 58. The genes were co-expressed with mL or cL        initially. In the case of the gH genes with loop choices for        CDR1 e.g. gH121, gH131, gH141 very little antibody was produced        in the culture supernatants. As no free light chain was detected        it was presumed that the antibody was being made and assembled        inside the cell but that the heavy chain was aberrant in some        way, possibly incorrectly folded, and therefore the antibody was        being degraded internally. In some experiments trace amounts of        antibody could be detected in ³⁵S labelling studies.    -   As no net antibody was produced, analysis of these constructs        was not pursued further.    -   When, however, a combination of the loop choice and the Kabat        choice for CDR1 was tested (mouse amino acids 26-35 inclusive)        and in which residues 31 (Ser to Arg), 33 (Ala to Thr), and 35        (Tyr to His) were changed from the human residues to the mouse        residue and compared to the first series, antibody was produced        for gH321, kgH331 and kgH341 when co-expressed with cL.        Expression was generally low and could not be markedly improved        by the insertion of the-Kozak consensus sequence 5′ to the ATG        of the signal sequence of the gene, as distinct from the case of        the gL genes where such insertion led to a 2-5 fold increase in        net antibody production. However, only in the case of gH341/mL        or kgH341/cL could marginal antigen binding activity be        demonstrated. When the kgH341 gene was co-expressed with        kgL221A, the net yield of antibody was too low to give a signal        above the background level in the antigen binding assay.

15.2.2. Framework Residues

-   -   As in the case of the light chain the heavy chain frameworks        were re-examined. Possibly because of the lower initial homology        between the mouse and human heavy variable domains compared to        the light chains, more amino acid positions proved to be of        interest. Two genes kgH341A and kgH341B were constructed, with        11 or 8 human residues respectively substituted by mouse        residues compared to gH341, and with the CDR2 residue 63        returned to the human amino acid potentially to improve domain        packing. Both showed antigen binding when combined with cL or        kgL221A, the kgH341A gene with all 11 changes appearing to be        the superior choice.

15.3 Interim Conclusions

-   -   It has been demonstrated, therefore, for OKT3 that to transfer        antigen binding ability to the humanised antibody, mouse        residues outside the CDR regions defined by the Kabat        hypervariability or structural loop choices are required for        both the light and heavy chains. Fewer extra residues are needed        for the light chain, possibly due to the higher initial homology        between the mouse and human kappa variable regions.    -   Of the changes seven (1 and 3 from the light chain and 6, 23,        71, 73 and 76 from the heavy chain) are predicted from a        knowledge of other antibody structures to be either partly        exposed or on the antibody surface. It has been shown here that        residues 1 and 3 in the light chain are not absolutely required        to be the mouse sequence; and for the heavy chain the gH341B        heavy chain in combination with the 221A light chain generated        only weak binding activity. Therefore the presence of the 6, 23        and 24 changes are important to maintain a binding affinity        similar to that of the murine antibody. It was important,        therefore, to further study the individual contribution of other        other 8 mouse residues of the kgH341A gene compared to kgH341.

16. Further CDR-Grafting Experiments

Additional CDR-grafted heavy chain genes were prepared substantially asdescribed above. With reference to Table 2 the further heavy chain geneswere based upon the kgH341 (plasmid pJA178) and gH341L (plasmid pJA185)with either mouse OKT3 or human KOL residues at 6, 23, 24, 48, 49, 63,71, 73, 76, 78, 88 and 91, as indicated. The CDR-grafted light chaingenes used in these further experiments were gL221, gL221A, gL221B andgL221C as described above. TABLE 2 OKT3 HEAVY CHAIN CDK GRAFTS 1. gH341and derivatives RES NUM 6 23 24 48 49 63 71 73 76 78 88 91 OKT3vhQ  K  A  I  G  F  T  K  S  A  A  Y gH341E  S  S  V  A  F  R  N  N  L  G  F JA178 gH341AQ  K  A  I  G  V  T  K  S  A  A  Y JA185 gH341EQ  K  A  I  G  V  T  K  S  A  G  G JA198 gH341*Q  K  A  I  G  V  T  K  N  A  G  F JA207 gH341*Q  K  A  I  G  V  R  N  N  A  G  F JA209 gH341DQ  K  A  I  G  V  T  K  N  L  G  F JA197 gH341*Q  K  A  I  G  V  R  N  N  L  G  F JA199 gH341CQ  K  A  V  A  F  R  N  N  L  G  F JA184 gH341*Q  S  A  I  G  V  T  K  S  A  A  Y JA203 gH341*E  S  A  I  G  V  T  K  S  A  A  Y JA205 gH341BE  S  S  I  G  V  T  K  S  A  A  Y JA183 gH341*Q  S  A  I  G  V  T  K  S  A  G  F JA204 gH341*E  S  A  I  G  V  T  K  S  A  G  F JA206 gH341*Q  S  A  I  G  V  T  K  N  A  G  F JA208 KOLE  S  S  V  A  R  N  N  L  G  F OKT3 LIGHT CHAIN CDR GRAFTS 2. gL221 andderivatives RES NUM 1  3  46 47 OKT3vl Q  V  R  W GL221 D  Q  L  L DA221gL22lA Q  V  R  W DA221A gL22lB Q  V  L  L DA221B GL221C D  Q  R  WDA221C RE1 D Q L  LMURINE RESIDUES ARE UNDERLINED

The CDR-grafted heavy and light chain genes were co-expressed in COScells either with one an there in various combinations but also with thecorresponding murine and chimeric heavy and light chain genessubstantially as described above. The resultant antibody products werethen assayed in binding and blocking assays with HPB-ALL cells asdescribed above.

-   -   The results of the assays for various grafted heavy chains        co-expressed with the gL221C light chain are given in FIGS. 7        and 8 (for the JA184, JA185, JA197 and JA198 constructs—see        Table 2), in FIG. 9 (for the JA183, JA184, JA185 and JA197.        constructs) in FIG. 10 (for the chimeric, JA185, JA199, JA204,        JA205, JA207, JA208 and JA209 constructs) and in FIG. 11 (for        the JA183, JA184, JA185, JA198, JA203, JA205 and JA206        constructs).    -   The basic grafted product without any human to marine changes in        the variable frameworks, i.e. gL221 co-expressed with gh341        (JA178), and also the “fully grafted” product, having most human        to marine changes in the grafted heavy chain framework, i.e.        gL221C co-expressed with gh341A (JA185), were assayed for        relative binding affinity in a competition assay against murine        OKT3 reference standard, using EPB-ALL cells. The assay used was        as described above in section 3.3. The results obtained are        given in FIG. 12 for the basic grafted product and in FIG. 13        for the fully grafted product. These results indicate that the        basic grafted product has negligible binding ability as compared        with the OKT3 murine reference standard; whereas the “fully        grafted” product has a binding ability very similar to that of        the OKT3 murine reference standard.    -   The binding and blocking assay results indicate the following:    -   The JA198 and JA207 constructs appear to have the best binding        characteristics and similar binding abilities, both        substantially the same as the chimeric and fully grafted gH341A        products. This indicates that positions 88 and 91 and position        76 are not highly critical for maintaining the OKT3 binding        ability; whereas at least some of positions 6, 23, 24, 48, 49,        71, 73 and 78 are more important.    -   This is borne out by the finding that the JA209 and JA199,        although of similar binding ability to one another, are of lower        binding ability than the JA198 and JA207 constructs. This        indicates the importance of having mouse residues at positions        71, 73 and 78, which are either completely or partially human in        the JA199 and JA209 constructs respectively.    -   Moreover, on comparing the results obtained for the JA205 and        JA183 constructs it is seen that there is a decrease in binding        going from the JA205 to the JA183 constructs. This indicates the        importance of retaining a mouse residue at position 23, the only        position changed between JA205 and JA183.    -   These and other results lead us to the conclusion that of the 11        mouse framework residues used in the gH341A (JA185) construct,        it is important to retain mouse residues at all of positions 6,        23, 24, 48 and 49, and possibly for maxim=binding affinity at        71, 73 and 78.    -   Similar Experiments were carried out to CDR-graft a number of        the rodent antibodies including antibodies having specificity        for CD4 (OKT4), ICAM-1 (R6-5), TAG72 (B72.3), and TNFα (61E71,        101.4, hTNF1, hTNF2 and hTNF3).

EXAMPLE 2 CDR-Grafting of a Murine Anti-CD4 T Cell Receptor Antibody,OKT4A

-   -   Anti OKT4A CDR-grafted heavy and light chain genes were        prepared, expressed and tested substantially as described above        in Example 1 for CDR-grafted OKT3. The CDR grafting of OKT4A is        described in detail in Ortho patent application PCT/GB 90 . . .        of even date herewith entitled “Humanised Antibodies”. The        disclosure of this Ortho patent application PCT/GB 90 . . . is        incorporated herein by reference. A number of CDR-grafted OKT4        antibodies have been prepared. Presently the CDR-grafted OKT4A        of choice is the combination of the grafted light chain LCDR2        and the grafted heavy chain ECDR10.

The Light Chain

-   -   The human acceptor framework used for the grafted light chains        was RE1. The preferred LCDR2 light chain has human to mouse        changes at positions 33, 34, 38, 49 and 89 in addition to the        structural loop CDRs. Of these changed positions, positions 33,        34 and 89 fall within the preferred extended CDRs of the present        invention (positions 33 and 34 in CDR1 and position 89 in CDR3).        The human to murine changes at positions 38 and 49 corresponds        to positions at which the amino acid residues are preferably        donor murine amino acid residues in accordance with the present        invention. A comparison of the amino acid sequences of the donor        murine light chain variable domain and the RH1 human acceptor        light chain variable further reveals that the murine and human        residues are identical at all of positions 46, 48 and 71 and at        all of positions 2, 4, 6, 35, 36, 44, 47, 62, 64-69, 85, 87, 98,        99 and 101 and 102. However the amino acid residue at position        58 in LCDR2 is the human RE1 framework residue not the mouse        OKT4 residue as would be preferred in accordance with the        present invention.

The Heavy Chain

The human acceptor framework used for the grafted heavy chains was KOL.

The preferred CDR graft ECDR10 heavy chain has human to mouse changes atpositions 24, 35, 57, 58, 60, 88 and 91 in addition to the structuralloop CDRs.

Of these positions, positions 35 (CDR1) and positions 57, 58 and 60(CDR2) fall within the preferred extended CDRs of the present invention.Also the human to mouse change at position 24 corresponds to a positionat which the amino acid residue is a donor marine residue in accordancewith the present invention. Moreover, the human to mouse changes atpositions 88 and 91 correspond to positions at which the amino acidresidues are optionally donor murine residues.

Moreover, a comparison of the marine OKT4A and human KOL heavy chainvariable amino acid sequences reveals that the murine and human residuesare identical at all of positions 23, 49, 71, 73 and 78 and at all ofpositions 2, 4, 6, 25, 36, 37, 39, 47, 48, 93, 94, 103, 104, 106 and107.

Thus the OKT4A CDR-grafted heavy chain HCDR10 corresponds to aparticularly preferred embodiment according to the present invention.

EXAMPLE 3 CDR-Grafting of an Anti-Mucin Specific Murine Antibody B72.3

The cloning of the genes coding for the anti-mucin specific murinemonoclonal antibody B72.3 and the preparation of B72.3 mouse-humanchimeric antibodies has been described previously (ret. 13 and WO89/01783). CDR-grafted versions of B72.3 were prepared as follows.

(a) B72.3 Light Chain

CDR-grafting of this light chain was accomplished by direct transfer ofthe murine CDRs into the framework of the human light chain RE1. Theregions transferred were: CDR Number Residues 1 24-34 2 50-56 3 90-96

-   -   The activity of the resulting grafted light chain was assessed        by co-expression in COS cells, of genes for the combinations:        -   272.3 cH/B72.3 cL    -   and B72.3 cH/B72.3 gL    -   Supernatants were assayed for antibody concentration and for the        ability to bind to microtitre plates coated with mucin. The        results obtained indicated that, in combination with the B72.3        cH chain, B72.3 cL and B72.3 gL had similar binding properties.

Comparison of the murine B72.3 and RE1 light chain amino acid sequencesreveals that the residues are identical at positions 46, 58 and 71 butare different at position 48.

Thus changing the human residue to the donor mouse residue at position48 may further improve the binding characteristics of the CDR-graftedlight chain, (B72.3 gL) in accordance with the present invention.

(b) B72.3 Heavy Chain

-   -   i. Choice of Framework        -   At the outset it was necessary to make a choice of human            framework. Simply put, the question was as follows: Was it            necessary to use the framework regions from an antibody            whose crystal structure was known or could the choice be            made on some other criteria?        -   For B72.3 heavy chain, it was reasoned that, while knowledge            of structure was important, transfer of the CDRs from mouse            to human frameworks might be facilitated if the overall            homology between the donor and receptor frameworks was            maximized. Comparison of the B72.3 heavy chain sequence with            those in Kabat (ref. 4) for human heavy chains showed            clearly that B72.3 had poor homology for KOL and NEWM (for            which crystal structures are available) but was very            homologous to the heavy chain for EU.

On this basis, EU was chosen for the CDR-grafting and the followingresidues transferred as CDRs. CDR Number Residues 1 27-36 2 50-63 3 93-102

-   -   -   Also it was noticed that the FR4 region of EU was unlike            that of any there human (or mouse) antibody. Consequently,            in the grafted heavy chain genes this was also changed to            produce a “consensus” human sequence. (Preliminary            experiments showed that grafted heavy chain genes containing            the EU PR4 sequence expressed very poorly in transient            expression systems.)

    -   ii. Results with Grafted Heavy Chain Genes        -   Expression of grafted heavy chain genes containing all human            framework regions with either gL or cL genes produced a            grafted antibody with little ability to bind to mucin. The            grafted antibody had about 1% the activity of the chimeric            antibody. In these experiments, however, it was noted that            the activity of the grafted antibody could be increased to            ˜10% of B72.3 by exposure to pHs of 2-3.5.        -   This observation provided a clue as to how the activity of            the grafted antibody could be improved without acid            treatment. It was postulated that acid exposure brought            about the protonation of an acidic residue (pKa of aspartic            acid=3.86 and of glutamine acid=4.25) which in turn caused a            change in structure of the CDR loops, or allowed better            access of antigen.        -   From comparison of the sequences of B72.3 (ref. 13) and EU            (refs. 4 and 5), it was clear that, in going from the mouse            to human frameworks, only two positions had been changed in            such a way that acidic residues had been introduced. These            positions are at residues 73 and 81, where K to E and Q to E            changes had been made, respectively.        -   Which of these positions might be important was determined            by examining the crystal structure of the KOL antibody. In            KOL heavy chain, position 81 is far removed from either of            the CDR loops.        -   Position 73, however, is close to both CDRs 1 and 3 of the            heavy chain and, in this position it was possible to            envisage that a K to E change in this region could have a            detrimental effect on antigen binding.

    -   iii. Framework Changes in B72.3 gH Gene        -   On the basis of the above analysis, E73 was mutated to a            lysine (K). It was found that this change had a dramatic            effect on the ability of the grafted Ab to bind to mucin.            Further the ability of the grafted B72.3 produced by the            mutated gH/gL combination to bind to mucin was similar to            that of the B72.3 chimeric antibody.

    -   iv. Other Framework Changes        -   In the course of the above experiments, other changes were            made in the heavy chain framework regions. Within the            accuracy of the assays used, none of the changes, either            alone or together, appeared beneficial.

    -   v. Other        -   All assays used measured the ability of the grafted Ab to            bind to mucin and, as a whole, indicated that the single            framework change at position 73 is sufficient to generate an            antibody with similar binding properties to B72.3.        -   Comparison of the B72.3 murine and EU heavy chain sequences            reveals that the mouse and human residues are identical at            positions 23, 24, 71 and 78.        -   Thus the mutated CDR-grafted B72.3 heavy chain corresponds            to a preferred embodiment of the present invention.

EXAMPLE 4 CDR-Grafting of a Murine Anti-ICAM-1 Monoclonal Antibody

A murine antibody, R6-5-D6 (BP 0314863) having specificity forIntercellular Adhesion Molecule 1 (ICAM-1) was CDR-grafted substantiallyas described above in previous examples. This work is described ingreater detail in co-pending application, British Patent Application No.9009549.8, the disclosure of which is incorporated herein by reference.

The human EU framework was used as the acceptor framework for both heavyand light chains. The CDR-grafted antibody currently of choice isprovided by co-expression of grafted light chain gL221A and graftedheavy chain gH341D which has a binding affinity for ICAM 1 of about 75%of that of the corresponding mouse-human chimeric antibody.

Light Chain

gL221A has marine CDRs at positions 24-34 (CDR1), 50-56 (CDR2) and 89-97(CDR3). In addition several framework residues are also the murine aminoacid. These residues were chosen after consideration of the possiblecontribution of these residues to domain packing and stability of theconfirmation of the antigen binding region. The residues which have beenretained as mouse are at positions 2, 3, 48 (?), 60, 84, 85 and 87.Comparison of the marine anti-ICAM 1 and human EU light chain amino acidsequences reveals that the murine and human residues are identical atpositions 46, 58 and 71.

Heavy Chain

gH341D has murine CDRs at positions 26-35 (CDR1), 50-56 (CDR2)and94-100B (CDR3). In addition murine residues were used in gH341D atpositions 24, 48, 69, 71, 73, 80, 88 and 91. Comparison of the marineanti-ICAM 1 and human EU heavy chain amino acid sequences are identicalat positions 23, 49 and 78.

EXAMPLE 5 CDR-Grafting of murine anti-TNFα antibodies

A number of marine anti-TNFα monoclonal antibodies were CDR-graftedsubstantially as described above in previous examples. These antibodiesinclude the marine monoclonal antibodies designated 61 E71, hTNF1, hTNF3and 101.4 A brief summary of the CDR-grafting of each of theseantibodies is given below.

61E71

A similar analysis as described above (Example 1, Section 12.1.) wasdone for 61E71 and for the heavy chain 10 residues were identified at23, 24, 48, 49, 68, 69, 71, 73, 75 and 88 as residues to potentiallyretain as murine. The human frameworks chosen for CDR-grafting of thisantibody, and the hTNF3 and 101.4 antibodies were RE1 for the lightchain and KOL for the heavy chain. Three genes were built, the first ofwhich contained 23, 24, 48, 49, 71 and 73 [gH341(6)] as murine residues.The second gene also had 75 and 88 as murine residues [gH341(8)] whilethe third gene additionally had 68, 69, 75 and 88 as murine residues[gH341(10)]. Each was co-expressed with gL221, the minimum grafted lightchain (CDRs only). The gL221/gH341(6) and gL221/gH341(8) antibodies bothbound as well to TNF as murine 61E71. The gL22l/gH341(10) antibody didnot express and this combination was not taken further.

Subsequently the gL221/gH341(6) antibody was assessed in an L929 cellcompetition assay in which the antibody competes against the TNFreceptor on L929 cells for binding to TNF in solution. In this assay thegL221/gH341(6) antibody was approximately 10% as active as murine 61E71.

hTNF1

hTNF1 is a monoclonal antibody which recognizes an epitope On humanTHF-. The EU human framework was used for CDR-grafting of both the heavyand light variable domains.

Heavy Chain

In the CDR-grafted heavy chain (ghTNF1) mouse CDRs were used atpositions 26-35 (CDR1), 50-65 (CDR2) and 95-102 (CDR3). Mouse residueswere also used in the frameworks at positions 48, 67, 69, 71, 73, 76,89, 91, 94 and 108. Comparison of the TNF1 mouse and EU human heavychain residues reveals that these are identical at positions 23, 24, 29and 78.

Light Chain

In the CDR-grafted light chain (gLhTNF1) mouse CDRs were used atpositions 24-34 (CDR1), 50-56 (CDR2) and 89-97 (CDR3). In addition mouseresidues wore used in the frameworks at positions 3, 42, 48, 49, 83, 106and 108. Comparison of the hTNF1 mouse and EU human light chain residuesreveals that these are identical at positions 46, 58 and 71.

The grafted hTNF1 heavy chain was co-expressed with the chimeric lightchain and the binding ability of the product compared with that of thechimeric light chain/chimeric heavy chain product in a TNF bindingassay. The grafted heavy chain product appeared to have binding abilityfor TNF slightly better than the fully chimeric product.

Similarly, a grafted heavy chain/grafted light chain product wasco-expressed and compared with the fully chimeric product and found tohave closely similar binding properties to the latter product.

hTDF3

hTNF3 recognizes an epitope an human TNF-α. The sequence of hTNF3 showsonly 21 differences compared to 61E71 in the light and heavy chainvariable regions, 10 in the light chain (2 in the CDRs at positions 50,96 and 8 in the framework at 1, 19, 40, 45, 46, 76, 103 and 106) and 11in the heavy chain (3 in the CDR regions at positions 52, 60 and 95 and8 in the framework at 1, 10, 38, 40, 67, 73, 87 and 105). The light andheavy chains of the 61371 and hTNF3 chimeric antibodies can be exchangedwithout loss of activity in the direct binding assay. However 61E71 isan order of magnitude less able to compete with the TNF receptor on L929cells for TNF-a compared to hTNF3. Based on the 61E71 CDR grafting datagL221 and gH341(+23, 24, 48, 49 71 and 73 as mouse) genes have beenbuilt for hTNF3 and tested and the resultant grafted antibody binds wellto TNF-a, but competes very poorly in the L929 assay. It is possiblethat in this case also the framework residues identified for OKT3programmed may improve the competitive binding ability of this antibody.

101.4

101.4 is a further marine monoclonal antibody able to recognize humanTNF-a. The heavy chain of this antibody shows good homology to KOL andso the CDR-grafting has been based on RE1 for the light chain and KOLfor the heavy chain. Several grafted heavy chain genes have beenconstructed with conservative choices for the CDR's (gH341) and whichhave one or a small number of non-CDR residues at positions 73, 78 or77-79 inclusive, as the mouse amino acids. These have been co-expressedwith cL or gL221. In all cases binding to TNF equivalent to the chimericantibody is seen and when co-expressed with cL the resultant antibodiesare able to compete well in the L929 assay. However, with gL221 theresultant antibodies are at least an order of magnitude 1 on able tocompete for TNF against the TNF receptor on L929 cells.

Mouse residues at other positions in the heavy chain, for example, at 23and 24 together or at 76 have been demonstrated to provide noimprovement to the competitive ability of the grafted antibody in theL929 assay.

A number of other antibodies including antibodies having specificity forinterleukins e.g. IL1 and cancer markers such as carcinoembryonicantigen (CRA) e.g. the monoclonal antibody A5B7 (ref. 21), have beensuccessfully CDR-grafted according to the present invention. It will beappreciated that the foregoing examples are given by way of illustrationonly and are not intended to limit the scope of the claimed invention.Changes and modifications may be made to the methods described whilststill falling within the spirit and scope of the invention.

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1. A cloning or expression vector containing a nucleic acid encoding an antibody molecule having affinity for a predetermined antigen and comprising a composite heavy chain and a complementary light chain, said composite heavy chain having a variable domain including complementary determining regions (CDRs) and framework regions, wherein said framework regions of said variable domain comprise predominantly human acceptor antibody heavy chain framework region residues, the remaining heavy chain framework region residues corresponding to the equivalent residues in a donor antibody having affinity for said predetermined antigen, wherein, according to the Kabat numbering system, in said composite heavy chain: said CDRs comprise donor residues at residues 31 to 35, 50 to 58, and 95 to 102; and said framework regions comprise donor residues at amino acid residues 6, 24, 48, 49, 71, 73, and
 78. 2. The cloning or expression vector of claim 1, wherein a residue selected from the group consisting of residues 1, 3, and 76 in said composite heavy chain are additionally donor residues.
 3. The cloning or expression vector of claim 1, wherein a residue selected from the group consisting of residues 36, 94, 104, 106, and 107 in said composite heavy chain are additionally donor residues.
 4. The cloning or expression vector of claim 3, wherein a residue selected from the group consisting of residues 2, 4, 38, 46, 67, and 69 in said composite heavy chain are additionally donor residues.
 5. The cloning or expression vector of claim 1, wherein amino acid residues 26 to 30 and 59 to 65 in said composite heavy chain are additionally donor residues.
 6. The cloning or expression vector of claim 1, wherein said complementary light chain is a composite light chain having a variable domain including complementary determining regions (CDRs) and framework regions, wherein said framework regions of said variable domain comprise predominantly human acceptor antibody light chain framework region residues, the remaining light chain framework region residues corresponding to the equivalent residues in a donor antibody having affinity for said predetermined antigen, wherein, according to the Kabat numbering system, in said composite light chain: said CDRs comprise donor residues at least at residues 24 to 34, 50 to 56, and 89 to 97; and amino acids residues 46, 48, 58, and 71 at least are donor residues.
 7. The cloning or expression vector of claim 6, wherein amino acid residues 1, 3, 60 (if this residue can form a salt bridge with residue 54), and 70 (if this residue can form a salt bridge with residue 24) in said composite light chain are additionally donor residues. 